Diagnostic assay for human matrix Gla-protein and its use as a biomarker

ABSTRACT

A diagnostic assay, preferably immunoassay, is provided for the detection and determination of MGP in a human serum sample, which comprises the use of one or more antibodies, in particular monoclonal antibodies, specifically recognising epitopes on and/or conformations of human Matrix Gla-Protein. Also, a method is provided for using MGP-related antigens as biomarkers for certain diseases, for example, atherosclerosis and other vascular diseases, and angiogenesis/neogenesis in tumor development. Further, monoclonal antibodies of class IgG are provided for use in the assay, which are defined herein as mAb3-15 and mAb35-49.

FIELD OF THE INVENTION

The present invention is in the field of molecular biology anddiagnostics. In particular, the invention relates to a diagnostic assayfor human Matrix Gla-protein (“MGP”), and its use as a biomarker forvascular condition and vascular new formation.

BACKGROUND OF THE INVENTION

Cardiovascular disease is one of the major life-threatening diseases inthe Western society, but biomarkers to monitor the severity or theprogression of the disease are presently not available. Also, the numberof biochemically detectable risk factors (e.g. serum cholesterol,triglycerides, ApoE genotype) is surprisingly low.

Vitamin K is a cofactor in the posttranslational conversion of glutamateresidues into y-carboxyglutamate (Gla). At this time 10 mammalianGla-containing proteins have been described in detail, and the number ofGla-residues per molecule varies from 3 (osteocalcin) to 13 (protein Z).In all cases in which their function was known, the activity of thevarious Gla-proteins was strictly dependent on the presence of theGla-residues (Shearer, M. J., Brit. J. Haematol. (1990) 75:156-162;Vermeer, C., Biochem. J. (1990) 266:625-636). Gla-proteins aresynthesized in various tissues, for instance the liver, bone and vesselwall. Blood coagulation factors 11 (prothrombin), VII, IX and X areexamples of Gla-proteins synthesized in the liver, examples of so-calledextrahepatic Gla-proteins are osteocalcin and Matrix Gla-Protein(Hauschka, P. V. et al., Phys. Rev. (1989) 69:990-1047).

Matrix Gla-Protein is a vitamin K-dependent protein synthesized in boneand in a number of soft tissues including heart and vessel wall. Inexperimental animals its soft tissue expression is high immediatelyafter birth, but decreases in the months thereafter. Only in cartillageand arteries its expression seems to continue lifelong. Although theprecise function of MGP on a molecular level has remained unknown sofar, experiments with MGP-deficient transgenic animals (“knock-out”mice) have shown that MGP has a prominent role in the prevention ofvascular mineralization: MGP-deficient animals were born to term butdeveloped severe aortic calcification (as analyzed by X-ray) in thefirst weeks of life; eventually all animals died within 6-8 weeks afterbirth due to rupture of the aorta or one of the other main arteries(Luo, G. et al., Nature (1997) 386:78-81).

MGP was discovered in bone (Price. P. A. et at., Biochem. Biophys. Res.Commun. (1983) 117:765-771), but in situ hybridization experimentsshowed that it is also expressed in other tissues including the vesselwall (Fraser, D. J. et al., J. BioL Chem. (1988) 263:11033-11036). Withpolyclonal antibodies raised against a synthetic peptide homologous tothe C-terminus of bovine MGP the protein was also found in cartilage viaimmunohistochemical staining (Loeser, R. F. et al., Biochem. J. (1992)282:1-6). A radioimmunoassay was developed for the detection of serumMGP in the rat, but in these experiments circulating MGP was correlatedwith maturation of rat bone, and not with vascular biology (Otawara, Y.et al, J. Biol. Chem. (1986) 261:10828-10832).

Research concerning the role of MGP in the vessel wall has not startedbefore the discovery by Luo et at (supra) that MGP is a strong inhibitorof vascular calcification in mice. Since then evidence has accumulatedsuggesting that bone calcification and atherosclerotic vessel wallcalcification proceed via very similar mechanisms, in which the sameproteins (including MGP) are used (Proudfoot, D. et al, Arterioscier.Thromb. Vasc. Biol. (1998) 18:379-388; Proudfoot, D. et al., J. Pathol.(1998) 185:1-3). Most studies on the regulation of MGP expression havebeen performed in smooth muscle cell cultures, with mRNA detection as ameasure for MGP synthesis. Recent studies in humans have shown that,although MGP mRNA is constitutively expressed by normal vascular smoothmuscle cells, it is substantially upregulated in cells adjacent to bothmedial and intimal calcification (Shanahan, C. M. et al., Cnt. Rev. inEukar. Gene Expr. (1998) 8:357-375).

Dhore, C et al., Faseb J. (1999) 13, p. A203, disclose that MGP isproduced in atherosclerotic plaques. The reference neither teaches norsuggests that MGP might enter the bloodstream from the plaque.

Sadowski, J. A. and O'Brien, M., Faseb J. (1991) 5, p. A944, disclosethe production of polyclonal antibodies to a vitamin K-dependent epitopeof MGP. The method is very general and can be used to any protein. Nospecific use of such antibodies for the assessment of MGP in human serumwas suggested.

Price, P. A. et al., Arteroscier. Thmmb. Vasc. Biol. (1998) 18:1400-1407disclose artificial calcification in a rat model system. This system isnot a model for atherosclerosis, because it is not associated withvascular inflammation and plaque formation. The conclusion obtained inthis model that vascular calcificsfion is associated with decreasedserum MGP levels, is opposite to the findings according to the presentinvention in atherosclerotic patients, i.e. increased serum MGP inatherosclerosis.

The prior art does neither teach or suggest the use of MGP as a markeror angiogenesis or cardiovascular disease, or the like, nor does itdisclose an assay for circulating human MGP.

As stated above, biomarkers to monitor the severity or the progressionof cardiovascular disease are not available up till now, and the numberof is biochemically detectable risk factors is very low. Therefore,there is clearly a need for biomarkers for vascular characteristics, forassessment of the severity or progression of atherosclerosis and relateddiseases, as well as for monitoring the effect of treatment duringvascular disease.

SUMMARY OF THE INVENTION

In accordance with an aspect of the present invention, the use of adiagnostic assay is provided for the detection and determination of MGPin a human serum sample, the assay comprising the use of one or moreantibodies, in particular monoclonal antibodies, specificallyrecognising epitopes on and/or conformations of human MatrixGla-Protein.

The invention further provides a method for the production of suchantibodies.

In another aspect of the invention, the assay comprises the use of saidantibodies for the detection of any Matrix Gla-Protein in human serum asthe total immunoreactive antigen, or for the detection of MatrixGla-Protein in human serum as the fraction of carboxylated MatrixGla-Protein (=5 Gla-residues/mol), or for the detection of MatrixGla-Protein in human serum as the fraction of under-carboxylated MGP (≦4Gla-residues/mol).

In still a further aspect of the invention, a method is provided forusing MGP-related antigens as biomarkers for certain diseases, forexample, atherosclerosis and other vascular diseases, andangiogenesis/neogenesis in tumor development.

In a preferred embodiment of the invention, monoclonal antibodies ofclass IgG are provided for use in said diagnostic immunoassay which areobtainable by hybridomas formed by fusion of cells from a mouse myelomaand spleen cells from a mouse previously immunized with a peptidehomologous to certain human MGP residues, in particular one of human MGPresidues 3-15, human MGP residues 35-49, and human MGP residues 54-84,which antibodies are also referred to herein as mAb3-15, mAb35-49, andmAb54-84, respectively. Of these, mAb3-15 and mAb35-49 are preferredantibodies.

These and other aspects of the present invention will be more fullyoutlined in the detailed description which follows.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 represents the assessed DNA nucleotide sequence and its derivedamino acid sequence of insert, e.g. MGP linked to murine dihydrofolatereductase (“DHFR”) and equipped with a N-terminal 6-His tag. The “Xasite” indicates a linker of -IIe-Glu-Gly-Arg- which is sensitive toproteolytic cleavage by clotting factor Xa. See also SEQ ID NOS: 1 and2.

FIG. 2 shows a dose-response reference curve for MGP in human referenceserum. Points are means of duplicate measurements made on 12 differentdays, error bars represent standard deviation.

FIG. 3 shows the species specificity of MGP-assay. A dose-response curvefor human serum MGP was compared with undiluted rat () and mouse (▭)serum. No response was obtained for the rodent sera.

FIG. 4 shows inter-individual variations in serum MGP levels. Fivevolunteers (30-50 year old males) were tested.

FIG. 5 shows blocking MGP antibodies with peptide 3-15. The stocksolution (100%) of peptides was 1.3 μg/ml. Peptide dilutions (∘) insteadof serum were incubated with antibodies and the excess of antibodies wasassessed with the microtiter plate assay. For comparison reference serumin various dilutions () was taken in the same run.

FIGS. 6-9 show serum MGP concentrations of patients suffering fromatherosclerosis, coronary atherosclerosis, diabetis mellitus andmalignancies, respectively, and of comparative groups of healthy people.These results are also summarized in Table 1.

FIG. 10 depicts the reactivity of mAb³⁻¹⁵ antibodies with purified rMGP(A) and rOC (B). The amount of recombinant protein on the microtiterplate was quantitated with anti-6His antibodies (), the reactivity withmAb³⁻¹⁵ was tested in the same plate (∘). In both cases staining wasperformed by incubation with a second antibody (rabbit anti-mouse totalIgG conjugated with horseradish peroxidase) as described below inMaterials and Methods.

FIG. 11 shows the absence of circadian pattern for circulating humanMGP. Points represent means ±SD of twelve different subjects; nine bloodsamples were obtained during the first 24 hours, and 5 samples wereobtained at 9 a.m. during the following two months.

FIG. 12 illustrates the antigen-capture technique: triplicatemeasurements of the standard reference curve were prepared on threeconsecutive days. Unknown plasma or serum samples can be read from thecurve. In this technique a labelled tracer peptide (MGP³⁻¹⁵) was addedto the serum, whereas monoclonal antibodies (mAb³⁻¹⁵) were coated ontothe microtiter plate. The apparent MGP concentrations in the test sampleare based on the assumption that the affinities of tracer and native MGPfor the antibody are similar.

FIG. 13 shows the means of triplicate MGP-measurements in pooled plasmaat various plasma dilutions using the sandwich ELISA technique. In thistechnique monoclonal antibodies against the MGP mid-sequence (mAb³⁵⁻⁴⁹)were coated onto the microtiter plate whereas biotinylated mAb³⁻¹⁵ wasused as a second antibody. Error bars represent SD.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on the surprising discovery that thevitamin K-dependent matrix Gla-protein, MGP, appears to play a key rolein preventing vascular calcification. MGP is synthesized in a vitaminK-dependent way in smooth muscle cells of the healthy vessel wall andits mRNA transcription is substantially upregulated in atheroscleroticlesions.

In order to investigate the potential value of MGP, or a functional partor derivative thereof, as a biomarker for vascular characteristics, MGPand fragments thereof were prepared synthetically and assays weredeveloped for the detection both of MGP in tissue, in particularvascular tissue, and of circulating MGP.

MGP, or functional fragments thereof, can be obtained in various ways,for example by recovering from a natural source, chemical synthesis, orusing recombinant DNA techniques.

In bone, MGP accumulates in relatively large quantities, which is whybone is the only tissue from which native MGP has been isolated thus far(Price, supra). However, under physiological conditions MGP originatingfrom human and bovine bone is one of the most insoluble proteins known.

The nucleotide and amino acid sequences of human MGP are shown in FIG.1. Comparison between its primary structure and the amino acid sequencederived from cDNA coding for MGP shows that in bone-derived MGP the last7 C-terminal amino acids are missing (Hale, J. E. et al., J. Biol.Chem., (1991) 266:21145-21149).

For the purpose of the present invention, synthetic peptides homologousto certain sequences of human MGP, for example to the MGP sequences3-15, 35-49, 63-75, and 54-84, were synthesized chemically and purified.Sequences as those mentioned above will be referred to hereinafter asMGP³⁻¹⁵, MGp³⁵⁻¹⁵, MGp⁶³⁻⁷⁵, and MGP⁵⁴⁻⁸⁴, respectively.

MGP can also be made using recombinant DNA techniques. When produced byrecombinant techniques, standard procedures for constructing DNAencoding the antigen, cloning that DNA into expression vectors,transforming host cells such as bacteria, yeast, insect, or mammaliancells, and expressing such DNA to produce the antigen may be employed.It may be desirable to express the antigen as a fusion protein toenhance expression, facilitate purification, or enhace solubility.

For example, first a DNA encoding the mature protein (used here toinclude any maturation form) is obtained. mRNA coding for MGP can beisolated from, e.g., cultured human osteoblasts and converted into thecorresponding cDNA using well known techniques. Alternatively, mRNAcoding for MGP can be obtained from other cell types, such aschondrocytes or smooth muscle cells. If the sequence is uninterrupted byintrons genomic or cDNA is suitable for expression in any host. If thereare introns, expression is obtainable in eukaryotic systems capable ofprocessing them. This sequence should occur in an excisable andrecoverable form. The excised or recovered coding sequence is thanplaced in operable linkage with suitable control sequences in areplicable expression vector. The vector is used to transform a suitablehost and the transformed host is cultured under favorable conditions toeffect the production of the recombinant protein. To improve theefficacy of prokaryotic MGP expression the cDNA may be equipped with anextension encoding a second protein, in such a way that expression willgive rise to a chimeric fusion protein.

Genomic or cDNA fragments are obtained and used directly in appropriatehosts, for example E. coli. The constructs for expression vectorsoperable in a variety of hosts are made using appropriate replicationand control sequences, as exemplified below. Suitable restrictions sitescan, if not normally available, be added to the ends of the codingsequence so as to provide an excisable gene to insert into thesevectors.

The control sequences, expression vectors, and transformation methodsare dependent on the type of host cell used to express the gene.Generally, prokaryotic, yeast and other eukaryotic cells (e.g. fungi,mammalian, insect, and plant cells) are presently available as hosts.Host systems which are capable of proper post-translational processingare preferred.

In one embodiment of the present invention MGP is suitably expressed asa chimeric protein linked to murine dihydrofolate reductase (“DHFR”) andequipped with a N-terminal 6-His tag for rapid purification; see FIG. 1and SEQ ID NOS: 1 and 2. The obtained 6-His tagged chimeric protein waspoorly soluble under physiological conditions. It is used for coatingthe microtiter plates in the assay which will be described below.Instead of DHFR, other proteins of sufficient size to efficientlyexpress a chimeric protein with MGP may be selected, known in the art,such as maltose binding protein.

Detection and quantitation of MGP in vascular tissue can be performed invarious ways. In a preferred embodiment of the present invention aimmuno-histochemical detection method is used employing one or moremonoclonal antibodies. The monoclonal antibody may suitably be human,mouse, rat or Camelidae (e.g. IIama) monoclonal antibody prepared byconventional methods, including those methods currently available forproducing monoclonal antibodies on a commercial scale, geneticallyengineered monoclonal antibody or antibodies fragments or antibodyproduced by in vitro immunisation by certain cells, and by phase displaytechniques. Also one or more polyclonal antibodies can be suitablyemployed, preferably antibodies generated generated in rabbit or goat.

The monoclonal antibodies are conveniently generated from mice which areimmunized with human MGP or a fragment thereof, for example MGP³⁻¹⁵,MGP³⁵⁻⁴⁹ or MGP⁵⁴⁻⁸⁴. Suitable and preferred monoclonal antibodiesagainst human MGP are obtained from the sera of, for example, Balb/Cmice which are immunized, e.g., with the peptide MGP³⁻¹⁵ or the peptideMGP³⁵⁻⁴⁹, using post-immune sera screening for their affinity towardpurified recombinant MGP, which was used as a chimeric construct withDHFR; see below.

Detection and quantitation of circulating MGP, which includesMGP-related antigen, can also be performed in various ways. Inaccordance with the present invention we have developed an ELISA-basedserum assay, a so-called “antibody-capture” assay in which the sampleMGP is complexed with an excess of purified monoclonal anti-MGPantibody, whereas in a second step the remaining unbound antibodies arequantified after binding to insolubilized recombinant MGP. Recordedimmunoreactive MGP turned out to be independent of sample preparationand showed small within-day and day-to-day fluctuations.

Besides the antibody capture technique and the competitive ELISAtechnique, which are both single-antibody ELISA assays, also thesandwich ELISA assay can be conveniently used for the detection andquantification of circulating MGP. depending on the availability of asuitable second antibody. Below we will first describe the developmentof the antibody-capture technique, followed by a more detaileddescription of the antigen-capture and sandwich ELISA techniques,respectively, as illustrated in FIGS. 12 and 13,

Dose-response curves were prepared with various dilutions of normalpooled serum and plasma. We found that these curves were reproducible intime with linear curves between 2- and 10-fold dilutions. Intra- andinter-assay variations in control samples were 12 and 15% and no crossreaction was observed with serum from rat and mice.

No circadian fluctuations were observed in a group of 6 men and 6 women(see FIG. 11). The range in the adult male population was found to bebetween 60 and 150% of the mean. MGP was independent of age in areference group of men and women >55 years old. Afterimmunohistochemical staining of vascular material a low but distinct MGPdeposition was observed in medial smooth muscle cells. Stronglyincreased deposition of MGP was observed in the center of the lipid corein advanced plaques, and in the border zones of mineral deposits.

Since it seems hard to envisage that a focal burst of MGP synthesiswould trigger calcification, it seems likely that MGP expression is aresponse to the precipitating mineral. Diabetics form a high risk groupfor developing media sclerosis, and therefore we have monitoredcirculating MGP in this patient group. It turned out that circulatingMGP concentrations in type I diabetics (n=12) were significantly higherthan in age- and sex-matched healthy controls, and the difference wasstatistically significant. In a group of 26 patients with coronaryatherosclerosis the serum MGP was even further increased. In a cohort of200 subjects no correlation was found between serum MGP and intimathickness (intima/media ratio). In subjects with postmenopausalosteoporosis or other metabolic bone disease serum MGP levels werewithin the normal range.

From the data obtained so far we conclude that our assay for serum MGPis reproducible, that arterial calcification is associated withincreased MGP deposition and that in advanced atherosclerosis theincreased MGP synthesis is reflected in its serum concentration. Apartfrom further elaboration and fine-tuning, which is well within the skillof ordinary skilled people who are familiar with the present inventionand the state of the art, the MGP assay is useful as a diagnostic toolfor the diagnosis or patient follow-up during the treatment ofatherosclerosis, and, possibly; related diseases.

The following is a typical example and preferred embodiment of theinvention illustrating the preparation of a monoclonal antibodyspecifcally recognising epitopes and/or conformations on human MatrixGla-protein, and various other aspects.

A. Preparation of Recombinant Human MGP

For cloning and expression of the chimeric construct of murinedihydrofolate reductase and MGP (DHFR-MGP) the commercially availableQIAexpress system was used (Qiagen Inc.). Fusion proteins wereconstructed using the pQE40 vector, wich contains an expression cassetteconsisting of the phage T5 promotor fused to the mouse DHFR protein. Tofacilitate protein purification by metal chelate affinity chromatographythe recombinant protein was engineered to contain a six residues longhistidine tail preceding the DHFR. For in frame fusion with the startcodon of the 6-His region the expression vector pQE40 was linearizedwith the restriction enzymes SphI and HindIII (Gibco BRL). Subsequently,the linearized vector was purified by agarose gel electrophoresisaccording to standard procedures.

For cloning of the human MGP cDNA, 5′-SphI and 3′-HindIII digestionsites were introduced by the polymerase chain reaction (PCR). PrimersMGPHUMT1 and MGPHUMT3 (for details see below) were used foramplification of cDNA coding for Ile-Glu-Gly-Arg-MGP. Each PCR reactionconsisted of 5 μl cDNA, 1 mM of each dNTP, 1 μg of each primer, 4 mMMgCl₂, 50 mM KCl, 10 mM Tris-HCl pH 8.3, 0.01% gelatin, and 2.5 U of Taqpolymerase (Pharmacia) in a total volume of 50 μl. Each PCR cycleconsisted of 1 min at 95° C., 2 min at 60° C. and 2 min at 72° C. Theamplified cDNA was digested with SphI and HindIII (Gibco-BRL) and theresulting fragments were then separated by agarose gel electrophoresisand isolated thereof by electro-elution. The isolated fragments andlinearized vector were then ligated in reactions containing 1 μldigested fragment, 1 μl digested pQE40 vector, (in a weight ratio of3:1) and subsequently transformed into E. coli strain M15[pREP4].Reaction conditions were: 1×T4 DNA ligase buffer, 1 mM ATP, and 1 WeissUnit of T4 DNA ligase (Pharmacia Biotech). Ligations were performedovernight at room temperature in a total volume of 10 μl. For theidentification of clones expressing a 6His tagged DHFR protein fused toMGP the colony blot procedure was used according to the QIAexpressionist manual.

Sequence analyses were performed on an ABI 310 genetic analyzer (PerkinElmer) according to the cycle sequencing procedure of the manufacturer.In brief, approximately 0.5 μg of CsCl₂ purified ds-DNA was added to areaction mixture which included 8 μl Terminator Ready Reaction Mix(Perkin Elmer) and 5 pmol of an appropriate primer in a total volume of20 μl adjusted with H₂O. Cycle sequencing was performed on a ThermoCycler 9600 (Perkin Elmer) according to the following protocol. Eachcycle consisted of 30 s at 95° C. , 15 s at 60° C., and 4 min at 72° C.After 25 cycles the procedure was terminated by a rapid ramp to 4° C.Subsequently, the products were purified on MicroSpin S-200 HR columns(Pharmacia Biotech) pre-equilibrated in H₂O according to themanufacturer's protocol. Finally, samples were precipitated withethanol, dried under vacuum and resuspended in 25 μl of TemplateSuppression Reagent (Perkin Elmer). Prior to loading the samples on theABI Prism 310 Genetic Analyser, samples were heated at 95° C. for 2 min.Sequence analysis of the samples was performed according the themanufacturer's manual.

All primers necessary for PCR and cycle sequencing were commerciallymanufactured by Perkin Elmer or Eurogentec with the following sequences:

-MGPHUMT1 (5′→3′), for SphI restriction site at 5′ end of human Xa-MGP:TAT GCA TGC ATT GAA GGT CGT TAT GAA TCA CAT GAA AGC (SEQ ID NO:3)-MGPHUMT3 (5′→3′), for HindIII restriction site at 3′ end of humanXa-MGP: TAT AAG CTT TTT GGC CCC TCG GCG CTT CCT (SEQ ID NO:4)

Characterization of Expression Product

All DNA fragments obtained through restriction enzyme digestion werepurified using the QlAquick PCR Purification Kit (Qiagen).

Ile-Glu-Gly-Arg-MGP cDNA was ligated into pQE40 and resulted in acorrect plasmid construct: pQE40-Ile-Glu-Gly-Arg-MGP as was establishedwith CsCl₂ purified DNA of the construct, sequenced on an ABI Prism 310sequencer. It was verified that the construct corresponded to pQE40Ile-Glu-Gly-Arg-MGP and that it was in the correct reading frame (seeFIG. 1; SEQ ID NO: 1). Calculated characteristics of the recombinantfusion protein 6His-DHFR-Ile-Glu-Gly-Arg-hMGP are: Mw=34.787 kD, 297aminoacid residues, pl=9.59. The resulting plasmid was transformed intoE. coli strain M15 [pREP4].

The construct was transformed into E. coli strain BL21 [DE3, pREP4],which is deficient in the Ion and ompT proteases. Selection ofrecombinant protein producing clones through the colony blot procedureand using the 6His monoclonal antibodies demonstrated several MGPproducing clones. From small-scale expression experiments followed byanalysis of the product by poly-acrylamide gel electrophoresis andWestern-blotting, cross reaction with 6His monoclonal antibodiesdemonstrated a specific binding against a protein with the expectedmolecular weight of approximately 35 kD and a minor band of 28 kD.

From the foregoing it can be concluded that good expression of theIle-Glu-Gly-Arg-MGP constructs as a part of a 6His DHFR fusion proteinin the pQE expression vector was accomplished in E. coli strain BL21[DE3, pREP4].

C. Preparation of Monoclonal Antibodies

Balb/C mice were immunised intraperitoneally either with a syntheticpeptide homologous to the N-terminus of human MGP (residues 3-15), or asynthetic peptide homologous to a mid-fragment of human MGP (residues35-49), or a synthetic peptide homologous to the C-terminus of human MGP(residues 54-84), which were coupled respectively to keyhole limpethemacin (KLH from Pierce, product nr. 77107). The antigen (20 μg) wasmixed with Freund's complete adjuvant and used for the firstimmunisation. The animals were boostered every second week with 20 μg ofantigen in Freund's incomplete adjuvant. Post-immune sera were screenedwith an indirect ELISA using purified recombinant6-His-DHFR-Ile-Glu-Gly-Arg-hMGP derived from E. coli, as describedbelow.

For the screening of post immune sera of mice and cell supematants anindirect ELISA was used in which the purified recombinant protein6-His-DHFR-Ile-Glu-Gly-Arg-hMGP was bound to the solid phase. The stocksolution of protein was diluted with sodium carbonate buffer (0.067 MNaHCO₃, 0.033 M Na₂CO₃, pH 9.6) to the required concentration (e.g. 50fold), and used to coat the wells of a 96-well microtiter plate (Costar,cat. no. 3590) by incubation for 1 hour at 37° C. After coating, theplates were washed three times with washing buffer (0.3 % Tween-20 inphosphate buffered saline) and 50 μl of either post-immune serum or cellsupematant were transferred into the coated microtiter plates andincubated for one h at 37° C. to achieve complete binding of theantibodies. Plates were washed three times with washing buffer andsubsequently incubated for one h at 37° C. with 50 μl of a solutioncontaining 1 μg/ml of rabbit anti mouse immunoglobulin conjugated tohorse radisch peroxidase (Dako, product nr. P0161) in washing buffer.Non-bound conjugated antibodies were removed by washing and 50 μl of TMBsubstrate (Roche) were transferred to each well; after 10 minutesreaction was stopped with 100 μl of H₂SO₄. Optical densities were readat 450 nm in an EIA reader (Bio-Rad, model 550). Post immune sere wasdiluted 100 times in 2% non fat dry milk (Protifar from NutriciaHolland) in phosphate buffered saline before use.

Spleen cells of good responding mice were fused with Sp 2/0 Ag 14myeloma cells. Cell suspensions were dispensed in ten 96-well microtiterplates and hybridomas were cultured for first 5 days in HAT selectivemedium containing RPMI 1640 medium (Gibco, cat. nr. 21060-017), 15%fetal clone serum (Hyclone-Greiner, cat. nr. A-6165), 1 mM pyruvate(Gibco, cat. nr. 11840-048), 2 mM L-glutamin (Serva, product nr. 22942),growth factor ESG (Costar), 1 mg/ml gentamycin (A.U.V. Cuijk Holland,cat. nr. VPK 62389) and 2% (v/v) 50×HAT. After 5 days half of the mediumwas refreshed with medium with same contents except that HAT wasreplaced by 2% (v/v) 50×HT (Gibco, cat. nr. 41065-012). After 11 daysthe cell supernatants of hybridoma cells were screened using the ELISAtechnique described above. Single cell cloning of positive hybridomacultures was performed by limiting dilution (see: Antibodies, alaboratory manual by E. Harlow and D. Lane, 1986, p222). This techniquewas repeated two more times for obtaining a monoclonal hybridoma cellline.

The antibody producing hybridoma clone was grown in roller bottle flaskwith 1 liter RPMI 1640 medium containing 3% fetal clone serum, 1 mMpyruvate, 2 mM L-glutamin and gentamycin at 37° C. for two weeks.Culture media were separated from the cells by centrifugation, and IgGwas isolated from the cell supernatants by protein-G sepharose columnchromatography (Pharmacia, cat. nr. 17-0618-02). Eluate fractions withA₂₈₀ >0.1 were pooled and concentrated using a concentrator filter witha 10 kD cut-off value. The yield was approximately 20 mg IgG per literof culture medium, the material obtained was stored at −20° C. at aconcentration of 1 mg/ml.

In a similar way antibodies against two other peptides, MGP³⁵⁻⁴⁹ andMGP⁵⁴⁻⁸⁴, were prepared.

D. Characterization of Antibodies

Antibodies were characterized by the Western blot technique, in whichtotal bacteria lysates were separated using an SDS-page minigelelectrophoresis apparatus (Bio-Rad). After electrophoresis had beencompleted, the proteins were transferred to a nitrocellulose membrane byblotting. Staining with the antibody resulted in a distinct protein bandat 35 kDa. This is the expected molecular weight of the fusion protein6His-DHFR-Ile-Glu-Gly-ArOsMGP. Monoclonal antibodies are of subclasstype IgG, as determined with-the mouse monoclonal antibody isotyping kit(Gibco. cat no. 10126-027). The monoclonal antibodies thus prepared willalso be referred to as mAb3-15, mAb35-49, and mAb54-84, respectively.Preferred monoclonal antibodies of the present invention are mnAb3-15and mAb35-49.

E. The Use of Antibodies in Assays for Circulating MGP

Three procedures for the MGP assay were worked out: the antibody-captureELISA and the competitive ELISA both enabe the detection of full lengthand fragmented MGP, and the sandwich ELISA, which allows the specificrecognition of intact molecules only.

E1. Procedure for the Antibody-capture ELISA.

In this assay a known excess of antibody is mixed with the test solutioncontaining an unknown amount of antigen, and the mixture is added to amicrotiter plate coated with antigen. Non-bound antibody from the testsample will be bound to the microtiter well, and is quantified with asecond (labelled) antibody.

Urea-solubilized recombinant 6His-DHFR-Ile-Glu-Gly-Arg-hMGP is diluted50 fold with coating buffer (0.1 M sodium carbonate buffer, pH 9.6) andused for coating an excess of 6His-DHFR-Ile-Glu-Gly-Arg-hMGP of themicrofiter plates (50 μl in each well), the plates are allowed to standfor 1 h at 37° C.;

Remaining active sites are blocked with blocking buffer (RocheDiagnostics Blocking reagent, cat nr. 1 112 589), 100 μl/well for 1 h at37° C.;

After repeated washing with washing buffer (0.3% (w/v) tween-20 inphosphate-buffered saline) the plates are ready for use;

Serum or plasma samples are supplemented with antibody solution (1 μg/mlin 3% (w/v) bovine serum albumine in phosphatebuffered saline) andincubated for 5 min at room temperature;

The samples (50 μl) are than transferred to the micmtiter plates andincubated for 1 h at 37° C.;

After washing 3 times with PBS/tween buffer (see above) the amount ofantibody bound to the plate is quantified using a second antibody:rabbit anti-mouse total IgG labeled with horse radish peroxidase (Dako,1 μg/mI in PBS-tween buffer) for TMBstaining (TMB enzymatic kit fromRoche);

After 15 min incubation the reaction is stopped by adding 1 M H₂SO₄whereafter the plate is read at 450 nm.

E2. Procedure for the Competitive ELISA.

In this assay a known amount of labelled antigen (the tracer) is mixedwith the test solution containing an unknown amount of antigen, and themixture is added tot the rnicrotiter-bound antibody. The antigen in thetest solution will compete with the tracer for binding to the antibodymatrix.

Biotinylation of tracer. A synthetic peptide analogous to the aminoacidsequence 3-15 in human MGP (MGP³⁻¹⁵) is used as a tracer. Biotin-X-NHS(Calbiochem # 2031188) is diluted in dimethyl sulphoxide (DMSO) to afinal concentration of 10 mg/ml and added to MGP³⁻¹⁵ (2 mg/ml in 0.1 MNa-borate buffer pH 8.5). Incubate at room temperature for 3 h in thedark. Remove the unbound biotin by overnight dialysis against phosphatebuffered saline (PBS) and store the labelled peptide in the dark at 4°C. after adding sodium zide to a final concentration of 0.1% (w/v).

Coating of microtiter plates. mAb3-15 antibodies (5 mcg/ml in carbonatebuffer, pH 9.6, 0.1 ml per well) are immobilized in wells of a highbinding 96-well microtiter plate (Coming #3590). After incubation for 1h at 37° C. remaining active sites are blocked with blocking reagent(Roche, 0.125 ml for 1 h at room temperature). All subsequent washingsteps between the various incubations are performed with 0.05% (v/v)tween-20 in 0.225 ml PBS.

Sample Preparation. Prepare the following sample tubes:

non-specific signal tube (blanc): 0.250 ml PBS/tween (0.05%, v/v)

total signal tube: 0.125 ml PBS-tween (0.05% v/v)

unknown sample tube: 0.120 ml PBS-tween (0.05%, v/v)+0.05 ml sample

standard tubes containing 0.125 ml of unlabelled MGP³⁻¹⁵ inconcentrations: 20, 10, 4, 2, 1, 0.4, and 0.2 nM.

Add 0.125 ml of tracer (0.2 nM in PBS/tween (0.05%, v/v) to all tubesexcept the non-specific signal tube and vortex. Transfer 0.1 ml of eachtube into a well of the abovementioned microtiter plate and incubate for1 h at 37° C.

Measurement. After washing, 0.1 ml of avidine-labelled horse radishperoxidase (0.1 mcg/mi) in PBS containing 10 mg/ml of bovine serumalbumin) is added. Incubate for 30 min at 37° C. After two additionalwashing steps with PBS (to remove all tween detergent), 0.1 ml offreshly prepared TMB substrate (TMB substrate kit, Pierce) is added toeach well. After 15 min incubation at room temperature the reaction isstopped by adding 0.1 ml of 2 N H₂SO₄, and the optical density ismeasured at 450 nm using an ELISA plate reader.

Calculation.

Subtract OD₄₅₀ of the non-specific signal tube from all others

Divide corrected OD₄₅₀ of standards and unknowns by OD of total signal:

A/A₀ (%)=OD of unknown or standard×100%/ OD of total signal

Make logistic curve fit with standards and interpolate MGP values ofunknown samples.

E3. Procedure for the sandwich ELISA (1).

In this assay two antibodies directed to different epitopes of oneantigen are used. One antibody is purified and bound to the solid phaseof a 96-well microtiter plate and the antigen in a test solution isallowed to bind. Unbound proteins are removed by washing, and thelabelled second antibody is allowed to bind to the antigen. Afterwashing, the assay is quantified by measuring the amount of labelledsecond antibody that is bound to the matrix. The procedure describedhere is for antibodies mAB35-49 and mAb3-15, but is applicable for otherantibodies as well.

Coating of microtiter plates. mAb35-49 antibodies (5 mcg/ml in carbonatebuffer, pH 9.6, 0.1 ml per well) are immobilized in wells of a highbinding 96-well microtiter plate (Corning #3590). After incubation for 1h at 37° C. remaining active sites are blocked with blocking reagent(Roche, 0.125 ml for 1 h at room temperature). All subsequent washingsteps between the various incubations are performed with 0.05% (v/v)Tween-20 in 0.225 ml PBS.

Sample preparation.

0.075 ml of unknown plasma are supplemented with 0.225 ml of wash buffer

reference samples are prepared by diluting pooled plasma wih wash bufferin concentrations of: 75%, 50%, 25%, 10%, 5%, 1%, 0.5% and 0.1%.

non-specific signal tube: 0.3 ml of wash buffer

transfer 0.1 ml of each sample to microtiter plate wells, incubate 1 hat 37° C.

Measurement. After washing, 0.1 ml of second antibody solution (5 mcg/mlbiotinylated mAb3-15) is added to each well and incubated for 1 h at 37°C. After washing 0.1 ml of Vectastain™ ABC reagent (Vector laboratories)is added to each well and incubated for 30 min at 37° C. After twoadditional washing steps with PBS to remove all tween detergent, 0.1 mlof freshly prepared TMB substrate (TMB substrate kit, Pierce) is addedto each well. After 15 min incubation at room temperature the reactionis stopped by adding 0.1 ml of 2 N H₂SO₄, and the optical density ismeasured at 450 nm using an ELISA plate reader.

Calculation.

Subtract the average OD₄₅₀ of the non-specific signal tube from allothers

Make logistic curve fit with standards and interpolate MGP values ofunknown samples.

E4. Procedure for the sandwich ELISA (2).

For the principle ot this test, see the introduction under E3, above.

Mouse anti-MGP3-15 (1 μg/ml in 0.1 M Na-carbonate buffer, pH 9.6) wascoupled to the microtiter plate by pipetting 50 μl of this solution perwell, and incubating the plates for 1 h at 37° C.;

Remaining active sites are blocked with blocking buffer (RocheDiagnostics Blocking reagent, cat nr. 1 112 589) 100 μl/well for 1 h at37° C.;

After repeated washing with washing buffer (0.3% (w/v) tween-20 inphosphate-buffered saline) the plates are ready for use: either serum orpurified MGP are pipetted in the well (50 μl), incubation for 60 min at37° C.;

Non-bound material is removed by washing with PBS-tween buffer;

The second antibody (mouse anti-MGP⁵⁴⁻⁸⁴, conjugated with horse-radishperoxidase) is pipetted (50 μl, 1 μg/ml), and the plates are incubatedfor 60 min at 37° C.;

After washing 3 times with PBS/tween buffer the amount of antibody boundto the plate is quantified using TMB-staining (TMB enzymatic kit fromRoche);

After 10 min incubation the reaction is stopped by adding 1 M H₂SO₄whereafter the plate is read at 450 nm.

F. Calibration Curve and Test Characteristics

An example of serum and plasma dilution curves obtained with theantibody capture ELISA is shown in FIG. 2. Calibration curves were madeon 12 different days using six different dilutions of pooled referenceserum. Each dilution was measured in duplicate, and the mean opticaldensities (OD) at 450 nm (SD) were expressed as a function of the serumconcentration (FIG. 2). At increasing dilutions of the serum sample,more anti-MGP was bound to the plate, with the buffer value as atheoretical maximum. The lower detection limit was defined as the meanOD+ three times the standard deviation of the buffer value, and amounted2.096−3×0.089=1.829, corresponding with an MGP concentration of 8.5 U/L.The intra- and inter-assay variation of the test were determined using afour-fold dilution of the reference serum. The intra-assay variation wascalculated by expressing the standard deviation as a percentage of themean obtained from 21 replicates, repeated on three different days andamounted 5.4%. For assessment of the inter-assay variation, duplicatemeasurements were made on 14 consecutive days after which the standarddeviation was expressed as a percentage of the means to give a value of12.6%.

In later experiments a gradual improvement can be seen, i.e. the buffervalues (no serum added) are somewhat higher, and the plateau values athigh MGP concentrations are lower. This depends on factors such asstaining time, etc.

G. Validity of the Test

Evidence for the validity of the test was obtained from its speciesspecificity: whereas a good response was obtained with human serum, ratand mouse serum gave no response at all (FIG. 3). This must be due tothe 2 amino acid residue difference between the rat and human MGPsequence 3-15.

Samples from five different volunteers were tested in duplicate, showinga good reproducibility and curves four of which were in the same range,whereas there was one outrider (FIG. 4).

For assessment of the normal range and reference groups, apparentlyhealthy subjects were recruited among the Maastricht population. The‘normal range’for MGP was established in 80 apparently healthy menbetween 20 and 84 years of age. It was found that the mean value forserum MGP in this group was 96±17 U/L. Hence the normal range (definedas the mean ±2 SD) was calculated to be between 62 and 130 U/L. Noapparent age-dependence was observed for MGP in this group. Similar datawere observed for elderly women (>60 years of age), but a larger rangewas found in women between 20-55 years. This may be related to hormonalchanges, and forms the basis for our decision that women <60 years oldwere not included in the experiments presented here.

The day-to-day and within-day variations were determined in a group of12 healthy men (20-35 years old), from whom blood was taken byvenipuncture on nine time points of one day, and on 4 different days at09.00 a.m. with one week intervals. The day-to-day and within-dayvariations were determined in a group of 12 healthy men (20-35 yearsold), from whom blood was taken by venipuncture on nine time points ofone day, and on 4 different days at 09.00 a.m. with one week intervals.The within-day variation was calculated for each subject separately byexpressing the standard deviation as a percentage of the mean of thenine time points, and amounted 11%. No distinct circadian pattern wasobserved. The day-to-day variation was calculated in a similar way fromthe four samples obtained with weekly intervals, and was found to be 8%.

The antibodies were tested further by incubating them with highlydiluted peptide MGP3-15. Even at concentrations as low as 0.09 μg/ml thebuffer value was almost completely blocked (FIG. 5).

H. Sample Preparation

To further evaluate the robustness of the assay, we have checked theinfluence of variations in the sample preparation at the followingsteps: centrifugation speed (1,500 and 10,000×g) during serumpreparation, centrifugation (10,000×g) after adding of mAb³⁻¹⁵,freeze-thawing of the serum sample (up to 8 cycles of freeze-thawing)and incubation time (between 3 and 60 minutes at room temperature) ofthe serum sample with mAb³⁻¹⁵. In none of these cases did the sampletreatment measurably affect the observed MGP concentration.

I. Assay Specificity

The mAb³⁻¹⁵ used in the assay was tested for its ability todifferentiate between two recombinant bone Gla-proteins: osteocalcin andMGP (both as chimeric constructs -linked with 6His-DHFR). Microtiterplates were coated with either purified recombinant MGP (1 μg/well) orequimolar amounts of purified recombinant osteocalcin. Couplingefficiency of both proteins was checked with anti-6His antibodies. As isshown in FIG. 10, both plates contained similar amounts of recombinantprotein (A: MGP; B: osteocalcin), and mAb³⁻¹⁵ reacted well with MGP, butnot with osteocalcin. The species specificity of mAb³⁻¹⁵ was testedfurther by comparing their reaction with human, rat, and murine serum.Cross reaction with rodent sera was below the detection limit (<8.5 U/L)in all dilutions tested.

J. Application and Potential Diagnostic Value of the MGP-assay

J1. Cardiovascular

In a group of over 500 non-hospitalized men between 40 and 60 years oldthe calcification of the abdominal aorta was measured by X-ray, and 28subjects with severe calcifications were selected. Circulating MGP wassignificantly higher in those with calcifications than in those in anapparently healthy age- and sex-matched reference population (p<0.0001,see FIG. 6).

J2. Coronary atherosclerosis

Patients with coronary atherosclerosis (n=26) were recruited from theUniversity Hospital., and their serum MGP was compared with that of thereference population. Serum MGP was significantly increased in thepatient group (p<0.001, see FIG. 7).

J3. Diabetes mellitus

Patients with type I diabetes mellitus (n=23) were recruited from theUniversity Hospital., and their serum MGP was compared with that of thereference population. Diabetes mellitus is a strong risk factor foratherosclerosis. Serum MGP was significantly increased in the patientgroup (p<0.01, see FIG. 8). Patients were selected independent on theduration of the disease.

J4. Malignancies

Patients with various malignancies (n=15) were recruited from theUniversity Hospital., and their serum MGP was compared with that of thereference population. Serum MGP was substantially and significantlyincreased in the patient group (p<0.0001, see FIG. 9). Malignancies willoften lead to angiogenesis (new formation of blood vessels).

J5. Other diseases

No correlations were found with early signs of atherosclerosis(increased intima-media thickness) nor with various diseases not relatedto the cardiovascular system (see Table 1). This suggests that the newlydeveloped assay is specific for diseases of the cardiovascular system,but many other patient groups have to be evaluated in this respect.

TABLE 1 Serum MGP concentrations in patients Serum MGP (% of age- andCondition Number sex-matched controls) High femur BMD (mean +> 1SD) 40 98.5 ± 5.7 Low femur BMD (mean −< 1SD) 38 102.0 ± 5.2 Senileosteoporosis 28 101.8 ± 6.8 Increased intima/media thickness 43   97.3 ±6.1 Data are given ± SE. Increased intima/media thickness was thehighest quartile from a group of 200 apparently healthy elderlysubjects. Subjects with high and low BMD of the femur neck were obtainedfrom a reference population (n = 250) recruited among the Maastrichtpopulation. Patients with osteoporosis, diabetes mellitus andatherosclerosis were obtained via various departments of the UniversityHospital Maastricht.

In still another aspect of the invention, a kit is provided comprising adevice suitable for carrying out the diagnostic assay according to thepresent invention, including one or more antibodies specificallyrecognising epitopes on and/or conformations of human MatrixGla-Protein, chemical agents useful or necessary to carry out the assay,which are familiar to the persons skilled in the art, and preferablyinstructions on how the assay is to be carried out in an optimal way.

The present disclosure is to be considered as in all respectsillustrative and not restrictive, the scope of the invention beingindicated by the appended claims, and all changes which come within themeaning and range of equivalency are intended to be embraced therein.

Hybridoma cell line B11A#1, which produces a monoclonal antibodydirected against an epitope in human matrix Gla protein residues 35-49,was deposited on Mar. 23, 2004 with Deutsche Sammlung vonMikroorganizmen und Zellkulturen GmbH (DSMZ) located at Mascheroder Weg1b, D-38124 Braunschweig, Germany as accession No. DSM ACC2639.Hybridoma cell line 52.1#1, which produces a monoclonal antibodydirected against an epitope in human matrix Gla protein residues 3-15,was deposited on Mar. 23, 2004 with DSMZ, as accession No. DSM ACC2638.

4 1 891 DNA Artificial Sequence Synthetic construct containing 6-Histag - DHFR - linker (=4 amino acids) - MGP 1 atg aga gga tcg cat cac catcac cat cac gga tcc ggc atc atg gtt 48 Met Arg Gly Ser His His His HisHis His Gly Ser Gly Ile Met Val 1 5 10 15 cga cca ttg aac tcg atc gtcgcc gtg tcc caa aat atg ggg att ggc 96 Arg Pro Leu Asn Ser Ile Val AlaVal Ser Gln Asn Met Gly Ile Gly 20 25 30 aag aac gga gac cta ccc tgg cctccg ctc agg aac gag ttc aag tac 144 Lys Asn Gly Asp Leu Pro Trp Pro ProLeu Arg Asn Glu Phe Lys Tyr 35 40 45 ttc caa aga atg acc aca acc tct tcagtg gaa ggt aaa cag aat ctg 192 Phe Gln Arg Met Thr Thr Thr Ser Ser ValGlu Gly Lys Gln Asn Leu 50 55 60 gtg att atg ggt agg aaa acc tgg ttc tccatt cct gag aag aat cga 240 Val Ile Met Gly Arg Lys Thr Trp Phe Ser IlePro Glu Lys Asn Arg 65 70 75 80 cct tta aag gac aga att aat ata gtt ctcagt aga gaa ctc aaa gaa 288 Pro Leu Lys Asp Arg Ile Asn Ile Val Leu SerArg Glu Leu Lys Glu 85 90 95 cca cca cga gga gct cat ttt ctt gcc aaa agtttg gat gat gcc tta 336 Pro Pro Arg Gly Ala His Phe Leu Ala Lys Ser LeuAsp Asp Ala Leu 100 105 110 aga ctt att gaa caa ccg gaa ttg gca agt aaagta gac atg gtt tgg 384 Arg Leu Ile Glu Gln Pro Glu Leu Ala Ser Lys ValAsp Met Val Trp 115 120 125 ata gtc gga ggc agt tct gtt tac cag gaa gccatg aat caa cca ggc 432 Ile Val Gly Gly Ser Ser Val Tyr Gln Glu Ala MetAsn Gln Pro Gly 130 135 140 cac ctt aga ctc ttt gtg aca agg atc atg caggaa ttt gaa agt gac 480 His Leu Arg Leu Phe Val Thr Arg Ile Met Gln GluPhe Glu Ser Asp 145 150 155 160 acg ttt ttc cca gaa att gat ttg ggg aaatat aaa ctt ctc cca gaa 528 Thr Phe Phe Pro Glu Ile Asp Leu Gly Lys TyrLys Leu Leu Pro Glu 165 170 175 tac cca ggc gtc ctc tct gag gtc cag gaggaa aaa ggc atc aag tat 576 Tyr Pro Gly Val Leu Ser Glu Val Gln Glu GluLys Gly Ile Lys Tyr 180 185 190 aag ttt gaa gtc tac gag aag aaa ggt tccaga tct gca tgc att gaa 624 Lys Phe Glu Val Tyr Glu Lys Lys Gly Ser ArgSer Ala Cys Ile Glu 195 200 205 ggt cgt tat gaa tca cat gaa agc atg gaatct tat gaa ctt aat ccc 672 Gly Arg Tyr Glu Ser His Glu Ser Met Glu SerTyr Glu Leu Asn Pro 210 215 220 ttc att aac agg aga aat gca aat acc ttcata tcc cct cag cag aga 720 Phe Ile Asn Arg Arg Asn Ala Asn Thr Phe IleSer Pro Gln Gln Arg 225 230 235 240 tgg aga gct aaa gtc caa gag agg atccga gaa cgc tct aag cct gtc 768 Trp Arg Ala Lys Val Gln Glu Arg Ile ArgGlu Arg Ser Lys Pro Val 245 250 255 cac gag ctc aat agg gaa gcc tgt gatgac tac aga ctt tgc gaa cgc 816 His Glu Leu Asn Arg Glu Ala Cys Asp AspTyr Arg Leu Cys Glu Arg 260 265 270 tac gcc atg gtt tat gga tac aat gctgcc tat aat cgc tac ttc agg 864 Tyr Ala Met Val Tyr Gly Tyr Asn Ala AlaTyr Asn Arg Tyr Phe Arg 275 280 285 aag cgc cga ggg gcc aaa aag ctt aat891 Lys Arg Arg Gly Ala Lys Lys Leu Asn 290 295 2 297 PRT ArtificialSequence Protein encoded by Sequence 1 containing 6-His tag - DHFR -linker (=4 amino acids) - MGP 2 Met Arg Gly Ser His His His His His HisGly Ser Gly Ile Met Val 1 5 10 15 Arg Pro Leu Asn Ser Ile Val Ala ValSer Gln Asn Met Gly Ile Gly 20 25 30 Lys Asn Gly Asp Leu Pro Trp Pro ProLeu Arg Asn Glu Phe Lys Tyr 35 40 45 Phe Gln Arg Met Thr Thr Thr Ser SerVal Glu Gly Lys Gln Asn Leu 50 55 60 Val Ile Met Gly Arg Lys Thr Trp PheSer Ile Pro Glu Lys Asn Arg 65 70 75 80 Pro Leu Lys Asp Arg Ile Asn IleVal Leu Ser Arg Glu Leu Lys Glu 85 90 95 Pro Pro Arg Gly Ala His Phe LeuAla Lys Ser Leu Asp Asp Ala Leu 100 105 110 Arg Leu Ile Glu Gln Pro GluLeu Ala Ser Lys Val Asp Met Val Trp 115 120 125 Ile Val Gly Gly Ser SerVal Tyr Gln Glu Ala Met Asn Gln Pro Gly 130 135 140 His Leu Arg Leu PheVal Thr Arg Ile Met Gln Glu Phe Glu Ser Asp 145 150 155 160 Thr Phe PhePro Glu Ile Asp Leu Gly Lys Tyr Lys Leu Leu Pro Glu 165 170 175 Tyr ProGly Val Leu Ser Glu Val Gln Glu Glu Lys Gly Ile Lys Tyr 180 185 190 LysPhe Glu Val Tyr Glu Lys Lys Gly Ser Arg Ser Ala Cys Ile Glu 195 200 205Gly Arg Tyr Glu Ser His Glu Ser Met Glu Ser Tyr Glu Leu Asn Pro 210 215220 Phe Ile Asn Arg Arg Asn Ala Asn Thr Phe Ile Ser Pro Gln Gln Arg 225230 235 240 Trp Arg Ala Lys Val Gln Glu Arg Ile Arg Glu Arg Ser Lys ProVal 245 250 255 His Glu Leu Asn Arg Glu Ala Cys Asp Asp Tyr Arg Leu CysGlu Arg 260 265 270 Tyr Ala Met Val Tyr Gly Tyr Asn Ala Ala Tyr Asn ArgTyr Phe Arg 275 280 285 Lys Arg Arg Gly Ala Lys Lys Leu Asn 290 295 3 39DNA Artificial Sequence Synthetic Primer 3 tatgcatgca ttgaaggtcgttatgaatca catgaaagc 39 4 30 DNA Artificial Sequence Synthetic Primer 4tataagcttt ttggcccctc ggcgcttcct 30

What is claimed is:
 1. A diagnostic kit for assaying human matrix Glaprotein in a serum sample, comprising: one or more monoclonal antibodiesdirected against an epitope in human matrix Gla protein residues 3-15 or3549, or combinations thereof, wherein said one or more antibodies areproduced by a hydridoma formed by fusion of cells from a mouse myelomaand spleen cells from a mouse previously immunized with a peptidehomologous to human matrix Gla protein residues 3-15 or 35-49.
 2. Amethod for determining matrix Gla protein present in a human serumsample, comprising: obtaining a human serum sample; exposing the humanserum sample to one or more monoclonal antibodies that specificallyrecognizes an epitope on human matrix Gla protein residues 3-15 or35-49, or a combination of monoclonal antibodies that specificallyrecognize an epitope on human matrix Gla protein residues 3-15 or 35-49,the one or more monoclonal antibodies being present in a predeterminedamount so that a portion of the one or more monoclonal antibodies remainuncomplexed to the human matrix Gla protein present in the sample; andmeasuring the amount of uncomplexed one or more monoclonal antibodies soas to determine the matrix Gla protein in the human serum sample.
 3. Aprocess for monitoring or detecting a disease, comprising: exposing ahuman serum sample to a monoclonal antibody of class IgG, comprising amonocolonal antibody directed against an epitope in human matrix Glaprotein residues 3-15 or 35-49, or a combination thereof, wherein saidantibody is produced by a hydridoma formed by fusion of cells from amouse myeloma and spleen cells tom a mouse previously immunized with apeptide homologous to human matrix Gla protein residues 3-15 or 35-49;and detecting a level of human matrix Gla protein in said serum sampleusing the monoclonal antibody, or a combination thereof, wherein anelevated level of human matrix Gla protein in said serum sample isassociated with coronary atherosclerosis, vascular calcificationangiogenesis, a disease of the vascular system, diabetes mellitus, orectopic calcification in tumor development, thereby monitoring ordetecting coronary atherosclerosis, vascula calcification angiogenesis,a disease of the vascular system, diabetes mellitus, or ectopiccalcification in tumor development.
 4. A monoclonal antibody of classIgG, comprising a monoclonal antibody directed against an epitope inhuman matrix Gla protein residues 3-15 wherein said antibody is producedby a hydridoma form by fusion of cells from a mouse mycloma and spleencells from a mouse previously immunized with a peptide bomologous tohuman matrix Gla protein residues 3-15.
 5. A monoclonal antibody ofclass IgG, comprising a monocolonal antibody directed against an epitopein human matrix Gla protein residues 35-49, wherein said antibody isproduced by a hydridoma formed by vision cell form a mouse myeloma andspleen cells from a mouse previously immunized with a peptide homologousto human matrix Gla protein residues 35-49.